U.S. patent number 11,150,186 [Application Number 17/348,819] was granted by the patent office on 2021-10-19 for protease transducers and sensors based on dna loops.
This patent grant is currently assigned to The Government of the United States of America, as represented by the Secretary of the Navy. The grantee listed for this patent is The Government of the United States of America, as represented by the Secretary of the Navy, The Government of the United States of America, as represented by the Secretary of the Navy. Invention is credited to Mario Ancona, Hieu Bui.
United States Patent |
11,150,186 |
Ancona , et al. |
October 19, 2021 |
Protease transducers and sensors based on DNA loops
Abstract
The stiffness and topology of ultra-small circular DNAs and
DNA/peptide hybrids are exploited to create a transducer of enzyme
activity with low error rates. The modularity and flexibility of
the concept are illustrated by demonstrating various transducers
that respond to either specific restriction endonucleases or to
specific proteases. In all cases the output is a DNA oligo signal
that, as we show, can readily be converted directly to an optical
readout, or can serve as input for further processing, for example,
using DNA logic or amplification. By exploiting the DNA hairpin (or
stem-loop) structure and the phenomenon of strand displacement, an
enzyme signal is converted into a DNA signal, in the manner of a
transducer. This is valuable because a DNA signal can be readily
amplified, combined, and processed as information.
Inventors: |
Ancona; Mario (Alexandria,
VA), Bui; Hieu (Alexandria, VA) |
Applicant: |
Name |
City |
State |
Country |
Type |
The Government of the United States of America, as represented by
the Secretary of the Navy |
Arlington |
VA |
US |
|
|
Assignee: |
The Government of the United States
of America, as represented by the Secretary of the Navy
(Washington, DC)
|
Family
ID: |
72749014 |
Appl.
No.: |
17/348,819 |
Filed: |
June 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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16848286 |
Apr 14, 2020 |
11067508 |
|
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62833953 |
Apr 15, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q
1/37 (20130101); C12Q 1/005 (20130101); G01N
33/542 (20130101); G01N 33/52 (20130101); G01N
21/6428 (20130101); G01N 2021/6432 (20130101) |
Current International
Class: |
G01N
21/64 (20060101); G01N 33/52 (20060101); G01N
33/542 (20060101); C12Q 1/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Seeman NC. Structural DNA nanotechnology: an overview. Methods Mol
Biol. 2005;303:143-166. doi:10.1385/1-59259-901-X:143. cited by
applicant .
Tyagi S, Kramer FR. Molecular beacons: probes that fluoresce upon
hybridization. Nat Biotechnol. 1996;14(3):303-308.
doi:10.1038/nbt0396-303. cited by applicant .
Yurke B, Turberfield AJ, Mills AP Jr, Simmel FC, Neumann JL. A
DNA-fuelled molecular machine made of DNA. Nature.
2000;406(6796):605-608. doi:10.1038/35020524. cited by applicant
.
Seelig G, Soloveichik D, Zhang DY, Winfree E. Enzyme-free nucleic
acid logic circuits. Science. 2006;314(5805):1585-1588.
doi:10.1126/science.1132493. cited by applicant .
Wu C, Cansiz S, Zhang L, et al. A Nonenzymatic Hairpin DNA Cascade
Reaction Provides High Signal Gain of mRNA Imaging inside Live
Cells. J Am Chem Soc. 2015;137(15):4900-4903.
doi:10.1021/jacs.5b00542. cited by applicant.
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Primary Examiner: Shen; Bin
Attorney, Agent or Firm: US Naval Research Laboratory
Roberts; Roy
Government Interests
FEDERALLY-SPONSORED RESEARCH AND DEVELOPMENT
This Application claims the benefit of U.S. Provisional Patent
Application No. 62/833,953 filed Apr. 4, 2019 and is a continuation
of U.S. patent application Ser. No. 16/848,286 filed Apr. 14, 2020,
now U.S. Pat. No. 11,067,508, the entirety of each of which is
incorporated herein by reference.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This Application claims the benefit of U.S. Provisional Patent
Application No. 62/833,953 filed Apr. 4, 2019 and is a continuation
of U.S. patent application Ser. No. 16/848,286 filed Apr. 14, 2020,
now U.S. Pat. No. 11,067,508, the entirety of each of which is
incorporated herein by reference.
Claims
What is claimed is:
1. An enzyme sensor system comprising: a loop transducer comprising
a stiffening domain of about 30 to 55 base pairs in length, a
cleavage domain cleavable by a protease, and a first hybridizing
domain of about 12 to 27 base pairs in length; and an output gate
comprising a second hybridizing domain of about 8 to 15 base pairs
in length and complementary to the first hybridizing domain, a
first fluorophore, and a quencher, wherein the system is configured
so that in the absence of the first hybridizing domain, the
quencher quenches the first fluorophore, and upon hybridization of
the two domains, the quencher become separated from the first
fluorophore sufficiently to allow fluorescence thereof.
2. The sensor system of claim 1, further comprising a second output
gate comprising a second fluorophore configured as a Forster
resonance energy transfer partner of the first fluorophore.
Description
BACKGROUND
Enzymes are protein catalysts vital to nearly all biology, allowing
nature to perform the myriad room-temperature or
near-room-temperature biochemical syntheses that make life
possible. A measure of the importance of these processes is that
enzymes constitute one quarter of the translation products of the
human genome (roughly 500 genes). Furthermore, enzymes are not only
key elements of healthy cell activities, but they are also crucial
for disease processes and can thus serve as markers for these
disease states. As a result, the ability to detect and monitor
enzymes such as proteases, esterases, kinases, etc. is a crucial
task in a multitude of applications in biology and medicine.
A wide range of enzyme detection systems exist for use in
applications in biomedicine, food, etc. Such methods, almost
entirely in vitro, can be grouped by the nature of their readouts
and the two main classes have optical and electrical outputs.
Performance and cost are the two main criteria, and new approaches
that have the potential to impact/improve in either of these areas
are always welcome. In addition, ideas that have the potential to
function in vivo, and can add significant sophistication are of
much interest for their potential to broaden capabilities.
A need exists for new techniques for detecting enzyme activity.
BRIEF SUMMARY
Described herein is a technique for enzyme detection/transduction
involving conversion to a DNA signal that can in turn be combined,
processed, and/or amplified using known DNA methods.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 schematically illustrates the concept of the nucleic acid
loop sensor/transducer.
FIG. 2 shows a loop sensor design for endonuclease.
FIG. 3A is a gel demonstrating the desired behavior of the HaeIII
loop sensor.
FIG. 3B shows photoluminescence data demonstrating confirming the
expected behavior of the HaeIII loop sensor while FIG. 3C shows the
behavior of the system over time.
FIGS. 4A-4C show operation of protease sensors.
FIGS. 5A-5D illustrate the operation of OR/NAND logical gates.
FIG. 6 shows DNA logic capable of implementing either OR or AND
with a photoluminescent output.
FIG. 7 illustrates a DNA amplification scheme.
DETAILED DESCRIPTION
Definitions
Before describing the present invention in detail, it is to be
understood that the terminology used in the specification is for
the purpose of describing particular embodiments, and is not
necessarily intended to be limiting. Although many methods,
structures and materials similar, modified, or equivalent to those
described herein can be used in the practice of the present
invention without undue experimentation, the preferred methods,
structures and materials are described herein. In describing and
claiming the present invention, the following terminology will be
used in accordance with the definitions set out below.
As used herein, the singular forms "a", "an," and "the" do not
preclude plural referents, unless the content clearly dictates
otherwise.
As used herein, the term "and/or" includes any and all combinations
of one or more of the associated listed items.
As used herein, the term "about" when used in conjunction with a
stated numerical value or range denotes somewhat more or somewhat
less than the stated value or range, to within a range of .+-.10%
of that stated.
Overview
While DNA is of course the basis of genetics, it has also given
rise to the field of DNA nanotechnology [1] wherein the information
content of the DNA (i.e., its base sequence) is used to program
form and/or function for a variety of potential non-biological
applications. As described herein, an enzyme signal is converted
into a DNA signal, in the manner of a transducer. This is valuable
because a DNA signal can be readily amplified, combined, and
processed as information. In doing so, two important concepts of
relevance are the DNA hairpin (or stem-loop) and strand
displacement. Both arise commonly in nature, and both are widely
exploited in DNA nanotechnology, e.g., as molecular beacons [2] and
for toehold-mediated strand displacement [3]. These capabilities
are also utilized jointly for purposes of DNA logic [4] and DNA
amplification [5]. The invention disclosed here makes use of all of
these ideas.
The subject invention is of this type in that the DNA is employed
as both a constructional and a computational material. From a
structural standpoint, the transducer is composed largely of DNA.
In this it takes particular advantage of two key characteristics of
DNA. One is its stiffness that arises from the base-stacking of
double-stranded (ds) DNA and that causes DNA to remain straight
when shorter than about 15 nm (coherence length). The other
relevant property of DNA is its helical nature which can impose
topological constraints on the ability of two strands of DNA to
hybridize.
A schematic of the loop sensor concept is shown in FIG. 1. As seen,
the basic design of the enzyme-to-DNA transducer consists of the
circular DNA or DNA/peptide structure plus an output gate that in
simplest form is a conventional molecular beacon. The circular
assembly is termed here a `loop transducer` and, as seen in the
figure, comprises three distinct functional domains. The first is a
stiffening domain wherein the blue loop strand has a complementary
DNA strand hybridized to it to form a duplex on one side of the
loop. Being double-stranded, this section is stiffened by the
well-known stacking interactions, and because its length is
typically at least half of the total, it keeps the entire loop open
with the remaining portions stretched in a `bow` configuration as
shown in FIG. 1. The second domain of the loop transducer is the
cleavage domain that contains the target of the enzyme. The
cleavage domain can be single- or double-stranded DNA, RNA, a
peptide, or combinations thereof. The third domain is the
hybridizing domain which is mostly or all single-stranded DNA that
is complementary to DNA in the output gate (on the right in FIG.
1). While the cleavage domain remains intact, the hybridization
domain is prevented from hybridizing to the output gate by (i) the
aforementioned stress in the loop and (ii) the topological
constraint inherent in intertwining two small loops. As a result,
if and when the enzyme cleaves the loop, it springs open, relieving
both the loop stress and the topological constraint, and thus makes
the DNA of the hybridizing domain available to infiltrate and open
the hairpin in the output gate by strand displacement. As a result,
the fluorophore and quencher pair at the ends of the output gate
become separated, and the fluorophore now provides a fluorescent
signal.
The released hybridizing domain constitutes the DNA output of the
loop transducer, and the conversion of this output to a fluorescent
signal by the molecular beacon represents the action of this
embodiment of the output gate (more complicated output gates are
described below). Within the concept just described there are many
aspects that can be varied and these may be regarded as of two
types, those made with functionality in mind and those associated
with optimization. While functionality is obviously of most
interest, achieving functionality requires optimization: for
example, if the hybridization strand is too short, then the hairpin
will never or almost never be open, and vice versa too long a
hybridization sequence will tend to leave the hairpin always or
almost always open.
A loop sensor design suitable for detecting endonucleases is shown
in FIG. 2. An endonuclease is a type of esterase that cleaves
(hydrolyzes) a phosphodiester bond in double stranded DNA (dsDNA)
at a specific location determined by the base sequence (with the
target and degree of specificity dependent on the particular
endonuclease). In this case, the entire structure is formed of DNA.
The longest strand (denoted LL) is typically 80 nucleotides long
but the length can be varied as desired (for example to within the
range of 60 to 100 or even more nucleotides). The loop is closed
with the addition of the stiffening strand (abbreviated as LS).
This leaves a nick in the loop that, as verified by molecular
dynamics simulation and by experiment, does not compromise the
stiffness of the loop transducer so long as the sequence is chosen
so that the stacking interaction at the nick is sufficient to
maintain the rigidity of the duplex [6]. Nevertheless, from the
perspective of susceptibility to melting or degradation (e.g., by
enzymatic attack if used in serum or in vivo), one may desire not
to have the nick, and it can be eliminated using a T4 ligase. The
molecular dynamics simulations were also used as check that the
stiffened designs were within the elastic limit of the dsDNA and
that buckling was unlikely to occur.
The cleavage domain can be varied to detect different substrates.
In the case of an endonuclease sensor, the target sequence should
be double-stranded, and this is done by the addition of the LC
strand seen in FIG. 2. For protease sensors, this cleavage domain
is the peptide substrate of the enzyme(s) of interest. Other
possible targets that could be incorporated in the cleavage domain
include mRNA or an RNA aptamer.
EXAMPLES
Nuclease Sensing
A loop sensor design suitable for detecting endonucleases was made
as shown in FIG. 2, with {LL}=80, {LS}=47, {LC}=10, {h_1}=8 and
{h_2}=8, and {c}=15, where the bracket notation denotes strand
length in nucleotides The loop is closed with the addition of the
stiffening strand (abbreviated as LS). This leaves a nick in the
loop that, as verified by molecular dynamics simulation and by
experiment, does not compromise the stiffness of the loop
transducer so long as the sequence is chosen so that the stacking
interaction at the nick is sufficient to maintain the rigidity of
the duplex. If desired, the nick can be eliminated using a ligase
such as T4 ligase.
Experiments were conducted to evaluate the optimal lengths of
sequences forming the transducer and output gate.
The first aspect of the design to be considered for optimization
was the hybridizing domain, in particular varying {h.sub.1} in the
range from 8 to 15 and {h.sub.2} in the range from 12 to 27. For
the experiments the target sequence in the cleavage domain was that
appropriate for HaeIII (GGCC) with the loop design kept fixed with
{LL}=80, {LS}=40, and {LC}=20 and only the output gate varied (and
simply shifting where the fixed sequences a.sub.2+h.sub.1 and
b.sub.1+h.sub.2 divide as {h.sub.1} and {h.sub.2} change). Using
gel electrophoresis, the various designs were assessed in the
presence or absence of the target enzyme. Remarkably, all of the
hybridizing domain designs worked well, in all cases showing low
levels of both false positives (in the absence of enzyme) and false
negatives (in the presence of enzyme). This means that there is
considerable flexibility in the design of the output gate and of
the hybridizing domain. Moreover, it demonstrates the robustness of
the overall design, and the effectiveness of the loop stresses and
topology in suppressing unwanted responses.
The second parameter considered for optimization was the length of
the stiffening LS strand; tested were lengths of 30, 40, 47, and
55. Fluorescence measurements were made to find the true (false)
positive rate from an estimate of the number of hairpins open
(closed) when the enzyme is present (absent). The best performing
designs have the highest true positive rate (TPR or sensitivity)
and the lowest false positive rate (FPR or one minus the
specificity). All of the tested stiffening strands give good
performance with the LS30 and LS47 designs being best, with the
former excelling in specificity and in maintaining a low FPR over
long periods of time, while the latter is preferred for sensitivity
and for the fastest response within the resolution of the
experiment, which was carried out for periods ranging from 2 to 21
hours. The general behavior is a rapid rise to a peak followed by a
slow degradation in performance, with again LS47 being best at
early times but LS30 performing better over longer times because of
its relative immunity to false positives. Finally, control
experiments were carried out in which the LS stand was either
missing entirely (with a ligated loop) or where it was such that
the dsDNA at the nick (in an unligated loop) could bend easily. In
both cases strong false positives were observed, with the hairpin
being opened by the hybridizing domain even when no endonuclease
was present. This shows that the stiffness of the loop is essential
to the proper functioning of the transducer.
For loop transducers that respond to endonucleases, another
variable to be considered for optimization is the length of the LC
strand that comprises the cleavage domain. The specific designs
examined had {LC} of 10, 20, and 40, with the hairpin labeled with
a donor (Cy3) and an acceptor (Cy5) dye. It was found that the
system functioned quite well with {LC}=20, but shows high false
positive rates when {LC}=10 and high false negative rates when
{LC}=40.
Based on the foregoing, an "optimal" design with {LL}=80, {LS}=30,
{LC}=20, {h.sub.1}=8, {h.sub.2}=8, and {.quadrature.}=15 was
investigated. Tests were made not just with the endonuclease of
interest but also with a different endonuclease (NcoI-HF, targeting
CCATGG instead of HaeIII's target GGCC, designed into the
transducer) to look for unwanted non-specific signals. FIG. 3A
presents results of polyacrylamide gel electrophoresis analysis.
Size standards appear in lanes 1 and 8. It was found that in the
absence of the target enzyme (HaeIII) and the output gate, the loop
transducer remained intact (lane 2), while in the presence of the
target enzyme it cleaved, and the associated increase in
electrophoretic mobility was observed in lane 3. The analogous
experiment with a non-target enzyme NcoI-HF showed no similar
transformation as observed in lane 4 (which was essentially
unchanged from the no-enzyme case of lane 2). Next introducing the
output gate, there was no change if the target enzyme was not
present (lane 5) or a non-target enzyme was present (lane 7), but
that the target enzyme instead cleaved the loop transducer (lane 6)
with some of the product as observed in lane 3 while the larger
fraction formed a slower moving complex resulting from the cleaved
loop transducer opening and hybridizing with the hairpin (intense
band in lane 5). This was all as desired.
The molecular beacon was used as the output gate to follow the
action spectroscopically with the data presented in FIG. 3B. This
assay was also attractive in allowing tracking of the temporal
evolution (FIG. 3C). With both the loop transducer and the output
gate present, no change was seen in fluorescence if either the
target enzyme was not present (upright triangles) or a non-target
enzyme (NcoI-HF) was present (inverted triangles); this is as
expected since under these conditions the loop transducer would not
be cleaved and thus could not activate the molecular beacon. By
contrast, in the presence of the target enzyme, the loop transducer
would be cleaved, the beacon would be activated, and a sharp
increase in fluorescence emission was indeed seen (circles) with
the reaction completing in about 40 minutes. This is near-ideal
transducer behavior.
As a test of the modularity of the design, a loop transducer
identical to that tested in FIGS. 3A-3C was prepared except that
the recognition sequence was replaced with the target of NcoI-HF
(CCATGG). Assessing its performance using a gel and fluorescence as
above, it was found to function quite well, responding strongly to
the presence of the target enzyme but not at all to a non-target
enzyme (HaeIII). This demonstrates the modularity of the design and
suggests that the approach could have broad applicability
Protease Sensing
Proteases are enzymes that cleave peptides, and therefore to create
a loop sensor for proteases a peptide segment is preferably be used
as the cleavage domain. For example, the protease trypsin targets
an eight-residue peptide which was incorporated into the DNA loop.
Before conducting physical experiments, a molecular dynamics
simulation was performed in order to look for any undesired
behavior, and especially for any unwanted interactions between the
peptide and the DNA. The simulation confirmed (over its 200 ns
duration) that the uncleaved loop transducer stayed in a stressed
`bow` configuration as depicted in FIG. 1, though of course there
were thermal fluctuations. Moreover, the peptide showed no
significant interaction with the DNA, and the peptide existed in a
stretched-out conformation that should be available for attack by
the protease.
The system behavior in the real world was examined using both a gel
and fluorescence with the results shown in FIGS. 4A (lanes 1-3) and
4B. The data shows that the transducer does indeed operate as
desired with the hairpin opened only when trypsin is present (lane
2) and not when it is either not present (lane 1) or when a
non-target protease, namely the tobacco etch virus TEV protease, is
present (lane 3). In addition, the transient spectroscopy in FIG.
4B shows more quantitatively that near-ideal sensor response is
realized, with little leakage when no trypsin is present and with
the full response to trypsin occurring in about 25 minutes. This
modularity of the basic sensor system design was enabled by the
non-specific mechanism of the loop stresses and topology that
suppress leakage.
As a second example of a protease transducer, a loop transducer was
produced that was identical to that described immediately above
except for having a cleavage domain for TEV protease (with target
sequence of seven peptides) rather than for trypsin. The data for
this transducer is shown in FIGS. 4A (lanes 4-6) and 4C. The gel
showed the desired behavior when no protease was present (lane 4)
and after TEV protease was added (lane 6). However, when a
non-target protease (trypsin) was added, two intense bands were
seen (lane 5) indicating that there is significant interaction of
the loop transducer and the output gate. This is also seen in the
transient fluorescence (FIG. 6C), though there is clearly a much
stronger response to TEV protease. It is possible that this
non-specific behavior of the TEV protease originates not in the
loop transducer but in the protease's star activity at the
non-optimal temperature of the experiment.
Logic
As shown herein, one can change target specificity of a system by
modifying the cleavage domain to convert the activity of various
enzymes into signals. An important benefit of performing this
conversion is that the products of such sensors are readily
combined to perform logical operations. This makes possible
information processing of multiple data streams, to improve the
reliability of the sensor output. Among other things, such designs
can in principle greatly reduce false positive rates, e.g., by
introducing an AND function that requires the presence of two
different nucleases in order to giving a positive response.
One can create a Boolean OR gate or a NAND gate, specifically by
creating two (or more) loop transducers with cleavage domains
appropriate to two (or more) target enzymes but sharing the same
sequence in their hybridizing domains that matches a particular
molecular beacon. This idea is illustrated in FIGS. 5A and 5B for a
2-input OR-gate that detects either or both HaeIII and NcoI-HF.
Upon sensing either or both enzymes, the corresponding loop
transducer(s) hybridized to the molecular beacon, resulting in an
increase in fluorescence as seen in FIG. 5C. In this design, the
output gate (labeled hairpin) was functionalized with a
donor-acceptor dye pair (Cye and Cy5), and opening of the hairpin
resulted in a rise in the donor (Cy3) emission. A NAND
functionality is achieved simply by monitoring the Cy5 dye instead.
FIG. 5D gives the emission of the Cy3 dye before and 2 hours after
the addition of either or both enzymes, and a clear increase in
emission is evident when either or both enzymes are present in the
solution. Interestingly, FIGS. 7C and 7D also show that the DNA
"circuit" acts not only as a binary OR gate, but as an analog adder
as well. This same type of approach can be applied to a protease
system.
One could feed the DNA output signals from multiple loop
transducers into DNA logic circuits, and thereby achieve a
modularity in which the transduction and logic functions are
separated. As illustration, we here consider realizing an AND
function on the outputs of two loop transducers using the design
shown in FIG. 6. When both DNA outputs from the loop transducers
are present, the labeled reporter duplex would self-assemble back
into a stabilized hairpin and in that state provide a fluorescent
output. Gel electrophoresis data showed sufficient results of the
AND-gate construction.
Further generalizations are possible using methods like those in
[4] and in other papers on DNA logic [8].
Amplification
Another use to which one can put the DNA output of the loop
transducer is amplification. The process takes advantage of the
catalytic capabilities of DNA oligonucleotides as exploited for
example in [5]. A depiction of what should readily be possible is
shown in FIG. 13.
Signal amplification is commonly used with sensors, typically by
introducing gain into the electrical readout circuitry. For
sensitive detection it is best to do this amplification as early as
possible following transduction so as to minimize the amplification
of noise. This suggests that for the loop transducers it would best
to do the amplification at the molecular level rather than in
subsequent optical or electrical stages. Two types of amplification
at the molecular level can be considered, with one being inherent
in the enzyme itself due to its turnover. The other is DNA
amplification either using enzyme-dependent methods such as the
polymerase chain reaction or rolling circle amplification (RCA), or
with a DNA-based scheme that is enzyme-free.
The output DNA signals from the loop converter system can benefit
from the integration of non-enzyme dependent amplification schemes.
Due to the high amplification range of the catalytic hairpin
assembly (CHA), the output DNA signals can be programmed as an
input/catalyst to trigger the amplification process of CHA systems.
A modified CHA amplification scheme for the proposed system was
tested in the absence of the endonuclease-to-DNA signal converter.
Fluorescence results indicate that the input signal was amplified
at least 4.times. times using the modified CHA scheme. Since the
modified CHA scheme only leveraged the internal-toehold mediate
strand displacement in order to be compatible with the existing
endonuclease-to-DNA signal converter, it was expected that the
amplification factor was less optimal compared to those systems
using the external-toehold mediated strand displacement. To improve
the amplification factor, the hairpin structures of the modified
CHA scheme were evaluated next. While maintaining the same
structure of the first hairpin, the second hairpin's stem was
padded with non-trivial bases to (i) increase its stability and
(ii) minimize leaks in the absence of the catalyst strand.
Fluorescence results indicate that the amplification factor
linearly improved as a function of additional non-trivial bases. It
is relevant that the proposed enzyme-to-DNA signal converter can be
equipped with an amplification circuit to boost the output signal
for low concentration detection applications.
Nucleic Acid Detection
Rather than as a detector of enzymes, the transducer can
alternatively be used to detect ssDNA or RNA. The idea is that the
oligo to be detected would hybridize to the cleavage domain of a
nuclease transducer, thus making the latter susceptible to attack
by an endonuclease and thereby revealing the presence of the target
oligo. To illustrate, we considered an RNA biomarker as the target,
with its binding to a loop transducer. In theory, any microRNA
biomarkers could be targeted in this way. Gel electrophoresis and
fluorescence spectroscopy confirmed the functionality of this
design, with the RNA-DNA hybrid indeed being cleaved by the HaeIII
endonuclease, thereby opening the loop and activating the molecular
beacon. It is expected that a wide variety of nucleic acids could
be detected in this way.
Further Embodiments
Beyond the above-demonstrated enzyme loop sensor effective for both
endonucleases and proteases, it should be possible to develop
similar schemes for other technologies such as microRNAs and
engineered proteins such as zinc-finger nucleases.
The use of a peptide nucleic acid (PNA)-based approach should be
possible either as the LS strand for increased rigidity and thermal
stability, or as a facile and lower cost means of inserting a
peptide into a DNA loop.
Beyond the demonstrated fluorescence outputs, color-change readouts
should also be possible, e.g., with the DNA release driving a
cross-linking reaction between particles (e.g., gold or magnesium).
Or another embodiment could involve tethering to metallic surfaces
so as to generate electrical outputs via standard electrochemical
methods.
Also contemplated are designs with the hybridized LS and/or LC
strands directly attached to the LL loop strand so that they would
not get lost and hence could be reconstituted from a dried
state.
The proposed system can be tethered to 2D substrates such as lipid
bilayers via the cholesterol-labeled DNA oligomer for enhancing
speed as well as utilizing the localization effect for sensing
surface-bound biomarkers.
Signals from this system should be measurable by, circular
dichroism (CD), UV-VIS, and excitonic-coupling phenomenon, in
addition to the techniques described in the examples.
Quenchers can be fluorescent dyes or other suitable quenchers of
fluorescence as known in the art.
Advantages
Described herein is a new technique for enzyme
detection/transduction by converting specific enzymatic activity
into a DNA signal that can in turn be combined, processed, and/or
amplified using known DNA methods. The advantages and new features
of the method over existing approaches may be summarized as
follows.
It provides a general technique that can be applied to
endonucleases and proteases, and potentially also to many other
classes of enzymes. This is made possible by the non-specificity of
the principle of operation (based on the loop stiffness and
topology) and by the demonstrated modularity of the design. These
considerations should make the approach broadly applicability to
many different areas in biomedicine, homeland security, etc.
The simplicity of the loop transducer design makes it scalable and
makes possible the processing of other information beyond
biomaterials.
With the loop transducer's simple DNA oligo output the technique
can be readily coupled to the world of DNA nanotechnology, and
especially to strand displacement networks. As illustrated in this
disclosure, two primary functions achievable in this way are
logical processing of the outputs and amplification of the
outputs.
The nano-size, non-toxic nature, and robustness to enzymatic attack
should make the approach adaptable to in vivo applications, unlike
many alternatives.
The approach is accomplished at very low cost in view of the
relative ease of obtaining the synthesized oligomers commercially.
In standard storage conditions, shelf-life should be excellent
given the known robustness of DNA.
The proposed system can withstand exonuclease digestion if using
the circular loop for in vivo application.
CONCLUDING REMARKS
All documents mentioned herein are hereby incorporated by reference
for the purpose of disclosing and describing the particular
materials and methodologies for which the document was cited.
Although the present invention has been described in connection
with preferred embodiments thereof, it will be appreciated by those
skilled in the art that additions, deletions, modifications, and
substitutions not specifically described may be made without
departing from the spirit and scope of the invention. Terminology
used herein should not be construed as being "means-plus-function"
language unless the term "means" is expressly used in association
therewith.
REFERENCES
[1] N. Seeman, Structural DNA Nanotechnology (Cambridge Univ.
Press, 2016). [2] S. Tyagi and F. R. Kramer, "Molecular beacons:
Probes that fluoresce upon hybridization," Nature Biotech. 14, 303
(1996). [3] B. Yurke, "A DNA-fueled molecular machine made of DNA,"
Nature 406, 605 (2000). [4] G. Seelig, D. Soloveichik, D. Y. Zhang,
and E. Winfree, "Enzyme-free nucleic acid logic circuits," Science
314, 1585 (2006). [5] C. Wu et al., "A nonenzymatic hairpin DNA
cascade reaction provides high signal gain of mRNA imaging inside
live cells," J. Am. Chem. Soc. 137, 4900-4903 (2015). [6] E.
Protozanova, P. Yakovchuk, and M. D. Frank-Kamenetskii,
"Stacked-unstacked equilibrium at the nick site of DNA," J. Mol.
Biol. 342, 775 (2004).
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